It is known that the enzyme neuraminidase is associated with the RNA viri of the myxovirus group which contains many highly infectious and pathogenic subgroups such as rubella, influenza, rabies, etc. The detection of the presence of this enzyme as an indicator of the presence of viri of the myxovirus group has been the subject of much work in recent years. It is well known that neuraminidase (hereinafter NASE) will cleave a ketosidic linkage between neuraminic acid as well as certain simple derivatives thereof and compounds which originally contained a phenolic or an alcoholic group, the oxygen atom of the phenolic or alcoholic group forming the ethereal oxygen of the ketosidic linkage. Methods were therefore devised to provide such ketosides and to detect the presence of NASE by testing the reaction mixture of the suspected sample and the ketosidic substrate for the presence of the phenol containing moiety. Several approaches to this problem have been developed. Most of them however depend upon the detection of a chromophoric moiety. Heretofore, the chromophoric moiety has been provided by the reaction of the liberated phenol with other chemical entities. Examples of this are to be found in the method disclosed by Tuppy, et al, for example, in FEBS Letters, 3, 72-75 (1969) and J. Virol., 6 556-558 (1970). An alternative is the chiobarbituric acid assay of sialic acids (J. Biol. Chem. 234, 1971-1975 (1959).
While these chromophoric methods are operative they leave much to be desired in the area of sinsitivity, and efficiency of operation. Conversion of the liberated alcoholic or phenolic moiety to a chromophore makes enzyme kinetic measurements time consuming, tedious, and costly. Thus, in screening work it would be desirable to detect the presence of viri of the myxovirus category quickly before clinical symptoms have appeared. In this, the incubation period of the disease, the virus is present in the system but often in rather small amounts. The presently available methods are not sensitive enough to detect the presence of the viri in the preclinical stages.
It would therefore be desirable to provide a NASE sensitive substrate which, upon reaction with NASE would provide a material detectable at extremely low concentrations since the production of such material is dependent upon the concentration of NASE which in turn is dependent upon the concentration of virus.
It would further be desirable to provide an efficient method of producing such a substrate.
Recent work in this area includes that of Privalova and Khorlin (Izvestiya Akademii Nauk SSR (Chemical Series) 2785 (1969)) in which the ketoside is formed between p-nitrophenol and the methyl ester of 2-chloro, 0- Tetracetyl-N-acetyl neuraminic acid utilizing silver carbonate as a catalyst. The NASE sensitive substrate was prepared by saponification of this acetylated ketoside to remove the 0-acetyl groups.
Conrow and Bernstein (J. Org. Chem., 36, 863 (1971)) found that certain steroids having a phenolic A-ring will readily form a glycosidic linkage with 0-tetraacetyl-2-bromoglucopyranose in the presence of a cadmium carbonate catalyst. It should be noted that the acetyl haloglucopyranose utilized by Conrow has a much simpler structure, especially in the steric sense than neuraminic acid or its derivatives. In particular, it should be noted that Conrow's material is not only devoid of an amino or amido group at the 5 position of the pyranose ring but, more importantly, is totally unsubstituted at the 2 position of the pyranose ring which, in Conrow's compound bears only the halo moiety, whereas in N-acetyl neuraminic acid that position bears a carboxy group which may be further converted into an ester grouping. Consideration of steric models demonstrate therefore that the 2 position of neuraminic acid is highly hindered and difficulties in reaction at that point might be expected.